Sorcin promotes migration in cancer and regulates the EGF-dependent EGFR signaling pathways.

The epidermal growth factor receptor (EGFR) is one of the main tumor drivers and is an important therapeutic target for many cancers. Calcium is important in EGFR signaling pathways. Sorcin is one of the most important calcium sensor proteins, overexpressed in many tumors, that promotes cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, malignant progression and resistance to chemotherapeutic drugs. The present work elucidates a functional mechanism that links calcium homeostasis to EGFR signaling in cancer. Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium-dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Of note, silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies.

Mitochondria inter-organelle relationships in cancer protein aggregation.

Mitochondria are physically associated with other organelles, such as ER and lysosomes, forming a complex network that is crucial for cell homeostasis regulation. Inter-organelle relationships are finely regulated by both tether systems, which maintain physical proximity, and by signaling cues that induce the exchange of molecular information to regulate metabolism, Ca2+ homeostasis, redox state, nutrient availability, and proteostasis. The coordinated action of the organelles is engaged in the cellular integrated stress response. In any case, pathological conditions alter functional communication and efficient rescue pathway activation, leading to cell distress exacerbation and eventually cell death. Among these detrimental signals, misfolded protein accumulation and aggregation cause major damage to the cells, since defects in protein clearance systems worsen cell toxicity. A cause for protein aggregation is often a defective mitochondrial redox balance, and the ER freshly translated misfolded proteins and/or a deficient lysosome-mediated clearance system. All these features aggravate mitochondrial damage and enhance proteotoxic stress. This review aims to gather the current knowledge about the complex liaison between mitochondria, ER, and lysosomes in facing proteotoxic stress and protein aggregation, highlighting both causes and consequences. Particularly, specific focus will be pointed to cancer, a pathology in which inter-organelle relations in protein aggregation have been poorly investigated.

Mitochondria: Insights into Crucial Features to Overcome Cancer Chemoresistance.

Mitochondria are key regulators of cell survival and are involved in a plethora of mechanisms, such as metabolism, Ca2+ signaling, reactive oxygen species (ROS) production, mitophagy and mitochondrial transfer, fusion, and fission (known as mitochondrial dynamics). The tuning of these processes in pathophysiological conditions is fundamental to the balance between cell death and survival. Indeed, ROS overproduction and mitochondrial Ca2+ overload are linked to the induction of apoptosis, while the impairment of mitochondrial dynamics and metabolism can have a double-faceted role in the decision between cell survival and death. Tumorigenesis involves an intricate series of cellular impairments not yet completely clarified, and a further level of complexity is added by the onset of apoptosis resistance mechanisms in cancer cells. In the majority of cases, cancer relapse or lack of responsiveness is related to the emergence of chemoresistance, which may be due to the cooperation of several cellular protection mechanisms, often mitochondria-related. With this review, we aim to critically report the current evidence on the relationship between mitochondria and cancer chemoresistance with a particular focus on the involvement of mitochondrial dynamics, mitochondrial Ca2+ signaling, oxidative stress, and metabolism to possibly identify new approaches or targets for overcoming cancer resistance.

Efficacy and Safety of Vitrectomy without Using Perfluorocarbon Liquids and Drainage Retinotomy Associated with Postoperative Positioning Based on Residual Subretinal Fluid for Rhegmatogenous Retinal Detachment.

Medical records of 75 eyes from 75 consecutive patients with uncomplicated rhegmatogenous retinal detachment (RRD) who underwent pars plana vitrectomy (PPV) were analyzed. Inclusion criteria were patients with RRD who underwent primary 23- or 25-gauge PPV with air, gas, or SiO tamponade and performed by a single surgeon, no use of perfluorocarbon liquids (PFCL) and drainage retinotomy, and follow-up ≥ six months. Exclusion criteria were patients who underwent previous vitreoretinal surgery, proliferative vitreoretinopathy (PVR) more than grade B, giant tears, and encircling band associated with PPV. The main endpoint was the anatomical retinal reattachment rate after a single surgical procedure. Secondary endpoints were best-corrected visual acuity (BCVA), postoperative retinal displacement, and intraoperative and/or postoperative complications. Primary anatomical success was achieved in 97.3% of cases using this modified surgical procedure. Retinal slippage occurred only in 28.2% of patients and it was not observed in all cases of macula-on RRD. The mean logMAR of the BCVA significantly improved in 92% of patients and no intraoperative complications were observed. The results suggest that complete subretinal liquid drainage is not mandatory for all RRD cases treated with PPV and that using PFCL and performing a drainage retinotomy are not essential in eyes with primary RRD and PVR less than grade B. Postoperative positioning after PPV for uncomplicated RRD based on the presence or absence of residual subretinal fluid at the end of surgery could limit the occurrence of postoperative retinal displacement, while promoting patient compliance.

Sorcin is an early marker of neurodegeneration, Ca2+ dysregulation and endoplasmic reticulum stress associated to neurodegenerative diseases.

Dysregulation of calcium signaling is emerging as a key feature in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), and targeting this process may be therapeutically beneficial. Under this perspective, it is important to study proteins that regulate calcium homeostasis in the cell. Sorcin is one of the most expressed calcium-binding proteins in the human brain; its overexpression increases endoplasmic reticulum (ER) calcium concentration and decreases ER stress in the heart and in other cellular types. Sorcin has been hypothesized to be involved in neurodegenerative diseases, since it may counteract the increased cytosolic calcium levels associated with neurodegeneration. In the present work, we show that Sorcin expression levels are strongly increased in cellular, animal, and human models of AD, PD, and HD, vs. normal cells. Sorcin partially colocalizes with RyRs in neurons and microglia cells; functional experiments with microsomes containing high amounts of RyR2 and RyR3, respectively, show that Sorcin is able to regulate these ER calcium channels. The molecular basis of the interaction of Sorcin with RyR2 and RyR3 is demonstrated by SPR. Sorcin also interacts with other ER proteins as SERCA2 and Sigma-1 receptor in a calcium-dependent fashion. We also show that Sorcin regulates ER calcium transients: Sorcin increases the velocity of ER calcium uptake (increasing SERCA activity). The data presented here demonstrate that Sorcin may represent both a novel early marker of neurodegenerative diseases and a response to cellular stress dependent on neurodegeneration.

Mitochondria as the decision makers for cancer cell fate: from signaling pathways to therapeutic strategies.

As pivotal players in cellular metabolism, mitochondria have a double-faceted role in the final decision of cell fate. This is true for all cell types, but it is even more important and intriguing in the cancer setting. Mitochondria regulate cell fate in many diverse ways: through metabolism, by producing ATP and other metabolites deemed vital or detrimental for cancer cells; through the regulation of Ca2+ homeostasis, especially by the joint participation of the endoplasmic reticulum in a membranous tethering system for Ca2+ signaling called mitochondria-ER associated membranes (MAMs); and by regulating signaling pathways involved in the survival of cancer cells such as mitophagy. Recent studies have shown that mitochondria can also play a role in the regulation of inflammatory pathways in cancer cells, for example, through the release of mitochondrial DNA (mtDNA) involved in the activation of the cGAS-cGAMP-STING pathway. In this review, we aim to explore the role of mitochondria as decision makers in fostering cancer cell death or survival depending on the tumor cell stage and describe novel anticancer therapeutic strategies targeting mitochondria.

Cancer metabolism and mitochondria: Finding novel mechanisms to fight tumours.

Mitochondria are dynamic organelles that have essential metabolic activity and are regarded as signalling hubs with biosynthetic, bioenergetics and signalling functions that orchestrate key biological pathways. However, mitochondria can influence all processes linked to oncogenesis, starting from malignant transformation to metastatic dissemination. In this review, we describe how alterations in the mitochondrial metabolic status contribute to the acquisition of typical malignant traits, discussing the most recent discoveries and the many unanswered questions. We also highlight that expanding our understanding of mitochondrial regulation and function mechanisms in the context of cancer cell metabolism could be an important task in biomedical research, thus offering the possibility of targeting mitochondria for the treatment of cancer.

Exogenous peptides are able to penetrate human cell and mitochondrial membranes, stabilize mitochondrial tRNA structures, and rescue severe mitochondrial defects.

Mutations in mitochondrial transfer RNA (mt-tRNA) genes are responsible for a wide range of syndromes, for which no effective treatment is available. We previously reported that transfection of the nucleotide sequence encoding for the 16-residue ß32_33 peptide from mitochondrial leucyl-tRNA synthetase ameliorates the cell phenotype caused by the mitochondrial tRNA mutations. In this work, we demonstrated that both the ß32_33 peptide linked with the known (L)-Phe-(D)-Arg-(L)-Phe-(L)-Lys (FrFK) mitochondrial penetrating sequence and, strikingly, the ß32_33 peptide per se, are able to penetrate both the plasma and mitochondrial membranes and exert the rescuing activity when exogenously administered to cells bearing the mutations m.3243A > G and m.8344A > G. These mutations are responsible for the most common and severe mt-tRNA-related diseases. In addition, we dissected the molecular determinants of constructs activity by showing that both the order of amino acids along the sequence and presence of positive charges are essential determinants of the peptide activity in cells and mt-tRNA structures stabilization in vitro. In view of future in vivo studies, this information may be required to design of ß32_33 peptide-mimetic derivatives. The ß32_33 and FrFK-ß32_33 peptides are, therefore, promising molecules for the development of therapeutic agents against diseases caused by the mt-tRNA point mutations.

Profiling calcium-dependent interactions between Sorcin and intrinsically disordered regions of human proteome.

BACKGROUND: Sorcin is a calcium sensor that exerts many calcium-related functions in the cells, e.g. it regulates calcium concentration in the cytoplasm, endoplasmic reticulum (ER) and mitochondria, by interacting with calcium pumps, exchangers and channels. Albeit Sorcin is an interesting potential cancer target, little is known about its interactors upon calcium-mediated activation. Our previous study suggested that Sorcin may recognize short linear binding motifs as the crystal structure revealed a self-interaction with a GYYPGG stretch in its N-terminus, and combinatorial peptide-phage display provided support for peptide-mediated interactions. METHODS: In this study we screened for motif-based interactions between Sorcin and intrinsically disordered regions of the human proteome using proteomic peptide phage display (ProP-PD). We identified a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a potential novel interactor and confirm the interaction through biophysical and cell-based approaches, and provide structural information through molecular dynamics simulations. RESULTS: Altogether, we identify a preferred motif in the enriched pool of binders and a peptide belonging to protein phosphatase 1 regulatory subunit 3G (PPP1R3G) as a preferred ligand. CONCLUSION: Through this study we gain information on a new Sorcin binding partner and profile Sorcin's motif-based interaction. GENERAL SIGNIFICANCE: The interaction between Sorcin and PPP1R3G may suggest a close dependence between glucose homeostasis and calcium concentration in the different cell compartments, opening a completely new and interesting scenery yet to be fully disclosed.

Roles of Sorcin in Drug Resistance in Cancer: One Protein, Many Mechanisms, for a Novel Potential Anticancer Drug Target.

The development of drug resistance is one of the main causes of failure in anti-cancer treatments. Tumor cells adopt many strategies to counteract the action of chemotherapeutic agents, e.g., enhanced DNA damage repair, inactivation of apoptotic pathways, alteration of drug targets, drug inactivation, and overexpression of ABC (Adenosine triphosphate-binding cassette, or ATP-binding cassette) transporters. These are broad substrate-specificity ATP-dependent efflux pumps able to export toxins or drugs out of cells; for instance, ABCB1 (MDR1, or P-glycoprotein 1), overexpressed in most cancer cells, confers them multidrug resistance (MDR). The gene coding for sorcin (SOluble Resistance-related Calcium-binding proteIN) is highly conserved among mammals and is located in the same chromosomal locus and amplicon as the ABC transporters ABCB1 and ABCB4, both in human and rodent genomes (two variants of ABCB1, i.e., ABCB1a and ABCB1b, are in rodent amplicon). Sorcin was initially characterized as a soluble protein overexpressed in multidrug (MD) resistant cells and named "resistance-related" because of its co-amplification with ABCB1. Although for years sorcin overexpression was thought to be only a by-product of the co-amplification with ABC transporter genes, many papers have recently demonstrated that sorcin plays an important part in MDR, indicating a possible role of sorcin as an oncoprotein. The present review illustrates sorcin roles in the generation of MDR via many mechanisms and points to sorcin as a novel potential target of different anticancer molecules.

The Role of Mitochondria in Inflammation: From Cancer to Neurodegenerative Disorders.

The main features that are commonly attributed to mitochondria consist of the regulation of cell proliferation, ATP generation, cell death and metabolism. However, recent scientific advances reveal that the intrinsic dynamicity of the mitochondrial compartment also plays a central role in proinflammatory signaling, identifying these organelles as a central platform for the control of innate immunity and the inflammatory response. Thus, mitochondrial dysfunctions have been related to severe chronic inflammatory disorders. Strategies aimed at reestablishing normal mitochondrial physiology could represent both preventive and therapeutic interventions for various pathologies related to exacerbated inflammation. Here, we explore the current understanding of the intricate interplay between mitochondria and the innate immune response in specific inflammatory diseases, such as neurological disorders and cancer.

The role of mitochondria-associated membranes in cellular homeostasis and diseases.

Mitochondria and endoplasmic reticulum (ER) are fundamental in the control of cell physiology regulating several signal transduction pathways. They continuously communicate exchanging messages in their contact sites called MAMs (mitochondria-associated membranes). MAMs are specific microdomains acting as a platform for the sorting of vital and dangerous signals. In recent years increasing evidence reported that multiple scaffold proteins and regulatory factors localize to this subcellular fraction suggesting MAMs as hotspot signaling domains. In this review we describe the current knowledge about MAMs' dynamics and processes, which provided new correlations between MAMs' dysfunctions and human diseases. In fact, MAMs machinery is strictly connected with several pathologies, like neurodegeneration, diabetes and mainly cancer. These pathological events are characterized by alterations in the normal communication between ER and mitochondria, leading to deep metabolic defects that contribute to the progression of the diseases.

Identification and binding mode of a novel Leishmania Trypanothione reductase inhibitor from high throughput screening.

Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against Leishmaniasis. This enzyme is fundamental for parasite survival in the host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize hydrogen peroxide produced by host macrophages during infection. In order to identify new lead compounds against Leishmania we developed and validated a new luminescence-based high-throughput screening (HTS) assay that allowed us to screen a library of 120,000 compounds. We identified a novel chemical class of TR inhibitors, able to kill parasites with an IC50 in the low micromolar range. The X-ray crystal structure of TR in complex with a compound from this class (compound 3) allowed the identification of its binding site in a pocket at the entrance of the NADPH binding site. Since the binding site of compound 3 identified by the X-ray structure is unique, and is not present in human homologs such as glutathione reductase (hGR), it represents a new target for drug discovery efforts.

Toward a Drug Against All Kinetoplastids: From LeishBox to Specific and Potent Trypanothione Reductase Inhibitors.

Leishmaniasis, Chagas disease, and sleeping sickness affect millions of people worldwide and lead to the death of about 50 000 humans per year. These diseases are caused by the kinetoplastids Leishmania, Trypanosoma cruzi, and Trypanosoma brucei, respectively. These parasites share many general features, including gene conservation, high amino acid identity among proteins, the presence of subcellular structures as glycosomes and the kinetoplastid, and genome architecture, that may make drug development family specific, rather than species-specific, i.e., on the basis of the inhibition of a common, conserved parasite target. However, no optimal molecular targets or broad-spectrum drugs have been identified to date to cure these diseases. Here, the LeishBox from GlaxoSmithKline high-throughput screening, a 192-molecule set of best antileishmanial compounds, based on 1.8 million compounds, was used to identify specific inhibitors of a validated Leishmania target, trypanothione reductase (TR), while analyzing in parallel the homologous human enzyme glutathione reductase (GR). We identified three specific highly potent TR inhibitors and performed docking on the TR solved structure, thereby elucidating the putative molecular basis of TR inhibition. Since TRs from kinetoplastids are well conserved, and these compounds inhibit the growth of Leishmania, Trypanosoma cruzi, and Trypanosoma brucei, the identification of a common validated target may lead to the development of potent antikinetoplastid drugs.

Not only P-glycoprotein: Amplification of the ABCB1-containing chromosome region 7q21 confers multidrug resistance upon cancer cells by coordinated overexpression of an assortment of resistance-related proteins.

The development of drug resistance continues to be a dominant hindrance toward curative cancer treatment. Overexpression of a wide-spectrum of ATP-dependent efflux pumps, and in particular of ABCB1 (P-glycoprotein or MDR1) is a well-known resistance mechanism for a plethora of cancer chemotherapeutics including for example taxenes, anthracyclines, Vinca alkaloids, and epipodopyllotoxins, demonstrated by a large array of published papers, both in tumor cell lines and in a variety of tumors, including various solid tumors and hematological malignancies. Upon repeated or even single dose treatment of cultured tumor cells or tumors in vivo with anti-tumor agents such as paclitaxel and doxorubicin, increased ABCB1 copy number has been demonstrated, resulting from chromosomal amplification events at 7q11.2-21 locus, leading to marked P-glycoprotein overexpression, and multidrug resistance (MDR). Clearly however, additional mechanisms such as single nucleotide polymorphisms (SNPs) and epigenetic modifications have shown a role in the overexpression of ABCB1 and of other MDR efflux pumps. However, notwithstanding the design of 4 generations of ABCB1 inhibitors and the wealth of information on the biochemistry and substrate specificity of ABC transporters, translation of this vast knowledge from the bench to the bedside has proven to be unexpectedly difficult. Many studies show that upon repeated treatment schedules of cell cultures or tumors with taxenes and anthracyclines as well as other chemotherapeutic drugs, amplification, and/or overexpression of a series of genes genomically surrounding the ABCB1 locus, is observed. Consequently, altered levels of other proteins may contribute to the establishment of the MDR phenotype, and lead to poor clinical outcome. Thus, the genes contained in this ABCB1 amplicon including ABCB4, SRI, DBF4, TMEM243, and RUNDC3B are overexpressed in many cancers, and especially in MDR tumors, while TP53TG1 and DMTF1 are bona fide tumor suppressors. This review describes the role of these genes in cancer and especially in the acquisition of MDR, elucidates possible connections in transcriptional regulation (co-amplification/repression) of genes belonging to the same ABCB1 amplicon region, and delineates their novel emerging contributions to tumor biology and possible strategies to overcome cancer MDR.

Binding of doxorubicin to Sorcin impairs cell death and increases drug resistance in cancer cells.

Sorcin is a calcium binding protein that plays an important role in multidrug resistance (MDR) in tumors, since its expression confers resistance to doxorubicin and to other chemotherapeutic drugs. In this study, we show that Sorcin is able to bind doxorubicin, vincristine, paclitaxel and cisplatin directly and with high affinity. The high affinity binding of doxorubicin to sorcin has been demonstrated with different techniques, that is, surface plasmon resonance, fluorescence titration and X-ray diffraction. Although the X-ray structure of sorcin in complex with doxorubicin has been solved at low resolution, it allows the identification of one of the two doxorubicin binding sites, placed at the interface between the EF5 loop the G helix and the EF4 loop. We show that Sorcin cellular localization changes upon doxorubicin treatment, an indication that the protein responds to doxorubicin and it presumably binds the drug also inside the cell, soon after drug entrance. We also demonstrate that Sorcin is able to limit the toxic effects of the chemotherapeutic agent in the cell. In addition, Sorcin silencing increases cell death upon treatment with doxorubicin, increases the accumulation of doxorubicin in cell nucleus, decreases the expression of MDR1 and doxorubicin efflux via MDR1.

Polyamine-trypanothione pathway: an update.

In trypanosomatids, polyamine and trypanothione pathways can be considered as a whole unique metabolism, where most enzymes are essential for parasitic survival and infectivity. Leishmania parasites and all the other members of the Trypanosomatids family depend on polyamines for growth and survival: the enzymes involved in the synthesis and utilization of spermidine and trypanothione, i.e., arginase, ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine synthase and in particular trypanothione synthetase-amidase, trypanothione reductase and tryparedoxin-dependent peroxidase are promising targets for drug development. This review deals with recent structure-based studies on these enzymes, aimed at the discovery of inhibitors of this pathway.

Small Molecules Targeting the miRNA-Binding Domain of Argonaute 2: From Computer-Aided Molecular Design to RNA Immunoprecipitation.

The development of small-molecule-based target therapy design for human disease and cancer is object of growing attention. Recently, specific microRNA (miRNA) mimicking compounds able to bind the miRNA-binding domain of Argonaute 2 protein (AGO2) to inhibit miRNA loading and its functional activity were described. Computer-aided molecular design techniques and RNA immunoprecipitation represent suitable approaches to identify and experimentally determine if a compound is able to impair the loading of miRNAs on AGO2 protein. Here, we describe these two methodologies that we recently used to select a specific compound able to interfere with the AGO2 functional activity and able to improve the retinoic acid-dependent myeloid differentiation of leukemic cells.

Surface Plasmon Resonance: A Useful Strategy for the Identification of Small Molecule Argonaute 2 Protein Binders.

Surface plasmon resonance (SPR) is one of the most important techniques for the detection and the characterization of molecular interactions. SPR technology is a label-free approach for monitoring biomolecular interactions in real time. The binding of analytes to molecules immobilized on a thin metal film (ligand) determines a change in the refractive index and, therefore in the angle of extinction of light, is reflected when polarized light hits the film, monitored in real time as a change in the position of the dip in reflected intensity. Since SPR detects mass, the technique is label-free.Here, we describe the use of SPR techniques to study the interaction between Argonaute 2 and small molecular compounds selected by means of high-throughput docking screening.

Short peptides from leucyl-tRNA synthetase rescue disease-causing mitochondrial tRNA point mutations.

Mutations in mitochondrial (mt) genes coding for mt-tRNAs are responsible for a range of syndromes, for which no effective treatment is available. We recently showed that the carboxy-terminal domain (Cterm) of human mt-leucyl tRNA synthetase rescues the pathologic phenotype associated either with the m.3243A>G mutation in mt-tRNA(Leu(UUR)) or with mutations in the mt-tRNA(Ile), both of which are aminoacylated by Class I mt-aminoacyl-tRNA synthetases (mt-aaRSs). Here we show, by using the human transmitochondrial cybrid model, that the Cterm is also able to improve the phenotype caused by the m.8344A>G mutation in mt-tRNA(Lys), aminoacylated by a Class II aaRS. Importantly, we demonstrate that the same rescuing ability is retained by two Cterm-derived short peptides, ß30_31 and ß32_33, which are effective towards both the m.8344A>G and the m.3243A>G mutations. Furthermore, we provide in vitro evidence that these peptides bind with high affinity wild-type and mutant human mt-tRNA(Leu(UUR)) and mt-tRNA(Lys), and stabilize mutant mt-tRNA(Leu(UUR)). In conclusion, we demonstrate that small Cterm-derived peptides can be effective tools to rescue cellular defects caused by mutations in a wide range of mt-tRNAs.